Generally, a natural type human growth hormone (hGH) consists of 191 amino acids having a molecular weight of about 21,500 dalton. The human growth hormone is secreted from the anterior pituitary and promotes the growth of bones and cartilages in vivo. Deficiency in growth hormone may cause short stature, increase the risk of cardiovascular diseases, decrease muscle and bone density, etc.
For the treatment of growth hormone deficiency, growth hormones derived from human pituitary gland or produced via genetic engineering technology, have been used since the late 1950s, and the global human growth hormone market had already reached about 3 trillion Korean Won in 2009. However, the above growth hormones had a drawback in that they had to be administered daily via subcutaneous injection and thus there has been a need for the improvement and increase of patient compliance. To comply with the request, long-acting human growth hormone has been developed, and in an effort to improve its long-acting property, sustained-release formulations, and engineering such as fusion to proteins or saccharides, glycotechnology, etc., have been employed. For example, Genentech Inc. released Nutropin Depot® as the first sustained-release human growth hormone product, and LG Life Sciences LTD. (Korea) released Declage Inj.® (KR Pat. Nos. 10-0236771 and 10-0329336) in 2007. Nevertheless, the sustained-release treatments still have problems, in that they cause unwanted immune responses in the body, induce pain due to the use of rather thick syringe needles, and have low production yield. In particular, Nutropin Depot® was withdrawn from the market due to its low efficacy compared to that of other first-generation products.
In order to improve the sustained activity of proteins other than sustained-release formulations, methods such as fusion to polyethylene glycol as a polymer (Sada et al., J. Ferment Bioeng 71, 137-139, 1991), glycoengineering (U.S. Pat. No. 7,217,689), and fusion to other proteins (WO93/15199) may be used to promote absorption, metabolism, and excretion in the body. However, the above methods have not been used as conventional half-life increase methods for various reasons, including low cost-effectiveness due to low production yield, induction of immune responses during long-term use, toxicity of chemical substances used during conjugation, etc. Accordingly, there has been a need for the development of a long-acting human growth hormone using a method of improving its long-acting property while minimizing the above-described problems. KR Patent No. 10-1183262 discloses the development of a long-acting human growth hormone protein in the form of a conjugate between a mutant of alpha-1 antitrypsin (A1AT) as a body protein and a human growth hormone attempting to improve half-life via protein size increase. Additionally, KR Patent Application Publication No. 10-2013-0029713 discloses a method of attempting to increase in vivo half life by adding N-glycan via at least one mutation on alpha-1 antitrypsin.
The alpha-1 antitrypsin (A1AT) mutant used by the present inventors is one where a particular amino acid is mutated in order to remove the intrinsic in vivo activity of the alpha-1 antitrypsin as a protease inhibitor while increasing its half-life. The protein sequence of alpha-1 antitrypsin which has its intrinsic activity as a protease inhibitor removed and a method of its preparation are disclosed in KR Patent Application Publication No. 10-2010-01165.
A long-acting human growth hormone is a fusion protein wherein a mutant of alpha-1 antitrypsin (A1AT) is fused to an N-terminus or C-terminus of a human growth hormone via genetic recombination, and is a material with improved half-life compared to those of first-generation human growth hormones. The long-acting human growth hormone was prepared so that it is expressed in CHO cells to be glycosylated while most of the first-generation human growth hormone drugs were manufactured in E. coli. 
The purification of alpha-1 antitrypsin was conventionally carried out by selective precipitation of impurities using polyglycol and pH (EP Patent No. 0097274) and cation-exchange resin (EP Patent Nos. 96929430 and 95112630). Additionally, the first generation human growth hormones were generally prepared by over-expression in E. coli in the form of an inclusion body, followed by refolding and purification with anion-exchange resin (KR Patent Nos. 1998-0003752, and Patra A K, Mukhopadhyay R, Mukhija R, Krishnan A, Garg L C, Panda A K. (2000) 18, 182-192, 2000; Protein Expr. Purif).
Meanwhile, the long-acting human growth hormone NexP-hGH is a novel material having a molecular weight of about 70 to 90 kDa, which is different from the first generation human growth hormones or alpha-1 antitrypsin in terms of physicochemical properties such as molecular weight, isoelectric point, glycan patterns, etc., and long-acting human growth hormone with high purity could not be obtained by the same/similar purification methods as those used for the first generation human growth hormones or alpha-1 antitrypsin. Furthermore, the purification method of long-acting human growth hormone has never been disclosed in patents or scientific papers.
Under the circumstances, the present inventors, while endeavoring to find an efficient purification method for the long-acting human growth hormone, the high purity of which has not been deemed possible, have discovered that high purity long-acting human growth hormone can be obtained via anion-exchange resin, hydrophobic resin, resin to which antibody fragments are attached, etc.
Additionally, the degree of influence by the glycosylation level of proteins present in the body on half-life and efficacies varies depending on the type of the proteins, for example, the conventionally used protein drugs erythropoietin and granulocyte colony-stimulation factor (GCSF). The in vivo efficacies of erythropoietin (J C Egrie and J K Browne, British J. of Cancer, 84, 3-10, 2001), a protein present in the body, vary depending on its sialic acid content. Specifically, erythropoietin has a high correlation between sialic acid content and in vivo efficacies, and highly glycosylated glycan containing a predetermined rate or higher of sialic acid content for the exhibition of appropriate efficacies. In contrast, the efficacies of GCSF showed a difference of up to 25% in in vitro efficacies depending on the presence/absence of glycans, but there was no difference in in vivo efficacies (H Bonig et al. Bone marrow transplantation, 28, 259-264, 2001).
As such, the present inventors, while endeavoring to develop a human growth hormone exhibiting long-acting pharmacological activities both in in vitro and in vivo conditions while having a lower risk of immunogenicity, discovered that an isoform of the NexP-hGH protein having a specific pI, among the above proteins produced in animal cells, has an excellent in vivo long-acting effect and growth effect, and thereby completed the present invention.
Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art. An objective of the present invention is to provide a long-acting human growth hormone NexP-hGH protein produced in animal cells, wherein an alpha-1 antitrypsin mutant (NexP) is fused with human growth hormone (hGH), having an isoelectric point (pI) of 5.2 or less.
Another objective of the present invention is to provide a method for preparing a long-acting human growth hormone NexP-hGH protein, the method comprising: (a) applying a biological fluid comprising a long-acting human growth hormone NexP-hGH protein, wherein an alpha-1 antitrypsin mutant (NexP) is fused with human growth hormone (hGH), to an affinity chromatography column filled with resin to which anti-alpha-1 antitrypsin antibody fragments are attached; and (b) separating long-acting growth hormone NexP-hGH protein having an isoelectric point (pI) of 5.2 or less according to the difference in isoelectric point (pI). A further objective of the present invention is to provide a method of purifying high purity long-acting human growth hormone NexP-hGH.